Diagnostic method for diabetes using C-13 labeled pyruvic acid

ABSTRACT

The present invention relates to a diagnostic agent for diabetes, which comprises glucose labelled with 13C at a specific position, or pyruvic acid labelled with 13C at least one specific position. According to the present invention, there is provided a diagnostic agent for diabetes which can be used safely without side effects to give accurate results immediately with less physical pains on the subject. The present diagnostic agent for diabetes can distinguish between healthy persons and patients with diabetes even under the circumstances where the patients are easily missed, and further it can determine the type of diabetes (insulin-dependent type or insulin-independent type).

This application is a divisional of U.S. Ser. No. 08/918,378, filed Aug.26, 1997, U.S. Pat. No. 5,916,538.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a diagnostic agent for diabetes and inparticular to a diagnostic agent for diabetes which comprises glucoselabelled with ¹³C at a specific position, or pyruvic acid labelled with¹³C at least one specific position.

2. Description of the Prior Art

Test methods generally used in the primary screening in diagnosis ofdiabetes are urine sugar test and fasting blood sugar levels test. Thesetests are simple and high in specificity, but are low in sensitivity andgive negative results for patients with light diabetes, so 70% or morepatients are missed and these tests are considered inadequate asscreening tests for diabetes (Sekikawa et al., Medical Practice 10:63,1993). On the one hand, a glucose tolerance test used for the diagnosisof diabetes brings about side effects due to administration of a largeamount of glucose, and this test requires the restraint of a subject forseveral hours and repeated collection of blood, and imposes heavyphysical burdens on the subject, and further the procedures aretroublesome, so this test is actually impossible to carry out as ascreening test of diabetes. Recently, blood HbA1C and fructosaminetests, which reflect average of blood sugar levels for a certain periodin the past, have been introduced as screening tests of diabetes in somefacilities. Under the existing circumstances, however, even those testsare cannot be said to be adequate in sensitivity and specificity forlight diabetes, and there remain the problem of a difference inmeasurement results among facilities.

Blood sugar level, HbA1C and fructosamine tests have been used widelyfor diagnosis of the type of diabetes, management of outpatients withdiabetes, and evaluation of therapeutic effects. However, blood sugarlevels would drop at the time of fasting in the case of light diabetes,while besides the above-described problems, the HbA1C and fructosaminetests have the problem that the results of the tests cannot be knownuntil a next visit to the hospital, so instructions would be given tothe patient on the basis of the past test results.

Under such circumstances, there is demand for developments in a testmethod which is effective for patients even with light diabetes andnon-invasive to the subjects and which give results immediately andaccurately for diagnosis of diabetes, management of patients withdiabetes and evaluation of therapeutic effects.

On the one hand, it is generally carried out to administer ¹³C-labeledglucose and measure ¹³C exhausting as carbon dioxide into an exhalationin order to assess energy expenditure. Because this analysis should beconducted under steady state, glucose should be administered for a longperiod before examination [J. J. Robert et al., J. Appl. Physiol. 63,1725-1732 (1987)]. Therefore, this analysis requires a long period forexamination and imposes the heavy pains on the subject, and is thuspractically not usable for diagnosis of diabetes.

It is reported that after naturally labelled ¹³C-glucose prepared fromC₄ plants is bolus administrated, the degree of exhalation of ¹³CO₂ isreduced in the case of patients with diabetes [P. Lefebvre, et al.,Diabetologia 14, 39-45 (1978); M. J. Arnaud, et al., Nutrition and theDiabetic Child, Pediat. Adolesc. Endocr. vol. 7, pp. 203-212, 1979].However, because naturally labelled ¹³C-glucose have 6 carbons randomlylabeled, we can scarecely evaluate an alternation of the metabolicpathway of glucose. Further, because the concentration of ¹³C innaturally labelled ¹³C-glucose is 2% or thereabout, it is necessary toadminister a large amount of glucose in order to monitor a change in theconcentration of CO₂ in an exhalation, and the burdens on the subjectare therefore heavy.

OBJECTS AND SUMMARY OF THE INVENTION

The object of the present invention is to provide a diagnostic agent fordiabetes to give accurate results immediately with few pains on thesubject.

As a result of their eager research, the present inventors found thatdiabetes and its type can be accurately diagnosed by administeringglucose labelled with ¹³C at a specific position or pyruvic acidlabelled with ¹³C at least one specific position, and then determiningdegrees of increase of ¹³C levels in exhaled CO₂, and they therebyarrived at the completion of the present invention.

That is, the present invention relates to a diagnostic agent fordiabetes comprising glucose labelled with ¹³C at a specific position.

The present invention further relates to a diagnostic agent for diabetescomprising pyruvic acid labelled with ¹³C at least one specificposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the main metabolic pathway of glucose for decarboxylation(numbers in the brackets next to CO₂ indicate the position of carbon inglucose).

FIG. 2 shows a method of sampling an exhalation from a rat.

FIG. 3 shows degrees of increase of ¹³C levels in exhaled CO₂ (Δ¹³C (‰))twenty minutes after intravenous injection of 1-³C-glucose (100 mg/kg).

FIG. 4 shows the relationship between the 1-¹³C-glucose breath test andfasting blood sugar levels.

FIG. 5 shows the relationship between the 1-¹³C-glucose breath test andthe total amount of secreted insulin during the first 15 min.

FIG. 6 shows the relationship between the 3-¹³C-glucose breath test andfasting blood sugar levels.

FIG. 7 shows the time course of ¹³C levels in exhaled CO₂ (Δ¹³C (‰))during the 3-¹³C-glucose breath test.

FIG. 8 shows degrees of increase of ¹³C levels in exhaled CO₂ (Δ¹³C (‰))from 10 to 20 minutes after administration of 3-¹³C-glucose.

FIG. 9 shows the relationship between the 3-¹³C-glucose breath test andfructosamine levels in blood.

FIG. 10 shows the time course of ¹³C levels in exhaled CO₂ (Δ¹³C (‰))during the 2-¹³C-glucose breath test.

FIG. 11 shows the time course of ¹³C levels in exhaled CO₂ (Δ^(—)C (‰))during the 6-¹³C-glucose breath test.

FIG. 12 shows the relationship between the 3-¹³C-pyruvate breath testand fasting blood sugar levels.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is described in detail.

The glucose in the present diagnostic agent for diabetes is glucoselabelled with ¹³C at a specific position, and the labelled position maybe any of positions 1 to 6.

Glucose labelled with ¹³C at a specific position includes e.g.commercial products such as 1-¹³C-glucose, 2-¹³C-glucose, 6-¹³C-glucose(produced respectively by EURISO-TOP Ltd., CIL Ltd., ISOTEC Ltd. andICON Ltd.), 3-¹³C-glucose (produced by CIL Ltd. and ICON Ltd.),4-¹³C-glucose (produced by CIL Ltd.) and 5-¹³C-glucose (produced by CILLtd.).

The pyruvic acid in the present diagnostic agent for diabetes is pyruvicacid labelled with ¹³ C at least one specific position.

The pyruvic acid in the present invention may be any pyruvic acid inwhich one, two or three of carbons at positions 1 to 3 have beenlabelled with ¹³C, preferably pyruvic acid labelled with ¹³C at position3. Specifically, commercial products such as sodium 3-¹³C-pyruvate(produced by ICON Ltd.) etc. can be used.

Because ¹³C is a stable isotope, there is no danger of exposure toradiation, and examinations can be effected safely.

In examinations using the present diagnostic agent for diabetes, ¹³Clevels (Δ¹³C (‰)) in exhaled CO₂ just after administration aredetermined followed by evaluation of data on degrees of increase of ¹³Clevels in exhaled CO₂ (Δ¹³C (‰)) at predetermined intervals (e.g. 5minutes, 20 minutes) after administration, or on time course (slope atthe start, change in the slope, peak time etc.) of degrees of increaseof ¹³C in exhaled CO₂ (Δ¹³C (‰)) for a predetermined time afteradministration. Although the sole evaluation by this breath test isuseful, the result of this test is preferably combined with blood sugarlevels, fructosamine levels etc. for synthetic judgment.

¹³C levels in exhaled CO₂ can be determined using gas chromatographymass spectrometry (GC-MS), infrared spectrophotometry, massspectrometry, photoacoustic spectrophotometry and NMR (nuclear magneticresonance).

The present diagnostic agent for diabetes can distinguish a group ofdiabetics from a normal group. In particular, the diagnostic agent fordiabetes containing glucose labelled with ¹³C can also distinguish thetype of diabetes (whether diabetes is insulin dependent or independent).

Further, it is possible to obtain a material for evaluation, whichdepending on a difference in the position of carbon labelled with ¹³C inglucose, is rendered special and advantageous to diagnosis of diabetes.

For example, glucose labelled with ¹³C at position 1 (1-¹³C-glucose) candistinguish between members with diabetes and healthy members in a groupwith normal fasting blood sugar levels, so this glucose is advantageousto the primary screening. Further, by virtue of its excellentrelationship to the total amount of insulin secreted, this glucose isused advantageously to determine a course of action for therapy. Glucoselabelled with ¹³C at position 3 (3-¹³C-glucose) can distinguish betweenpatients with insulin-dependent diabetes and patients withinsulin-independent type in the case of almost the same blood sugarlevels, so this glucose is used advantageously for diagnosis of the typeof diabetes. Moreover, this glucose may distinguish between theinsulin-dependent diabetes and insulin-independent diabetes in the caseof similar fructosamine levels, so it may be advantageously used forknowing an alternation in the disease (transition from the independenttype to dependent type), which can be easily missed when evaluation ismade using only fructosamine levels.

As shown in FIG. 1, carbons in glucose are decarboxylated in differentmetabolic pathways depending on their positions. Therefore, in caseswhere glucose labelled with ¹³C at a specific position has been given,we can evaluate an alternation in the glucose metabolic pathways bydetermining the degree of exhalation of CO₂.

The present diagnostic agent for diabetes is manufactured intopharmaceutical preparations such as parenteral agents (tablets,capsules, powder, granules, liquid etc.), injections etc., depending onthe administration route, by solely using glucose labelled with ¹³C at aspecific position (referred to hereinafter as labelled glucose) orpyruvic acid labelled with ¹³C at least one specific position (referredto hereinafter as labelled pyruvic acid) or by mixing it with fillers orcarriers. The fillers or carriers may be any of those conventionallyused in this field if they are pharmaceutically acceptable. The type andcomposition of such preparations are altered suitably according to theroute and method of administration. For example, water is used as aliquid carrier. As solid carriers, cellulose derivatives such ashydroxypropyl cellulose and organic acid salts such as magnesiumstearate etc. are used. Water, physiological saline and various buffersolutions are generally desirable in the case of injections. Suchpreparations may be lyophilized for use as oral medicines, or thelyophilized preparations may be dissolved in suitable injection solventse.g. liquids for intravenous administration, such as sterilized water,physiological saline, electrolyte etc. just before use.

The content of the labelled glucose or labelled pyruvic acid in thepharmaceutical preparation varies according to the type ofpharmaceutical preparation, and is usually in the range of 10 to 100% byweight, preferably 50 to 100% by weight. In the case of injections, forexample, the substituted glucose or substituted pyruvic acid is addedusually in an amount of 1 to 40% by weight. In the case of capsules,tablets, granules and powder, the content of the substituted glucose orsubstituted pyruvic acid is in the range of about 10 to 100% by weight,preferably 50 to 100% by weight, with the remainder being carriers.

The present diagnostic agent for diabetes should be administered at sucha dosage as to enable confirmation of an increase of ¹³C levels in anexhalation after administration. Depending on the age and weight of thepatient and the object of breath test, the dosage for eachadministration ranges from about 1 to 2000 mg/kg body weight in the caseof an adult.

Hereinafter, the present invention is described in more detail byreference to Examples, which however are not intended to limit the scopeof the invention.

EFFECT OF THE INVENTION

According to the present invention, there is provided a diagnostic agentfor diabetes which can be used safely without side effects and giveaccurate results immediately with less physical burdens on the subject.The present diagnostic agent for diabetes can not only distinguishbetween healthy persons and patients with diabetes even under thecircumstances where the patients are easily missed, but can alsodetermine the type of diabetes (insulin-dependent type orinsulin-independent type).

PREFERRED EMBODIMENTS OF THE INVENTION Test Example

[1] Materials and Methods

(1) Animals

Male Sprague-Dawley strain (SD) rats were purchased from Nippon CharlesRiver K.K. Neonatal rats were purchased along with a lactating rat. Therats were bred at 23° C.±2° C. under 55±10% humidity before use. Therats were fed standard diet and water ad libitum.

(2) Generation of diabetic rats

For insulin-dependent diabetes, insulin-deficient type diabetes wasgenerated in a matured rat by intraperitoneally administering ofstreptozotocin (STZ) (“Saibokogaku” (Cell Engineering), Extra Issue,Medical Experiment Manual Series, Strategy for Study of Diabetes, editedby Susumu Kiyono and Yoshikazu Oka, published by Shushunsha, Japan).

STZ (No. S-0130, a product of Sigma) was administered intraperitoneallyat a dose of 90 mg/kg to the matured rat previously fasted overnight.Two days later, blood was collected from the tail vein, and its bloodsugar level was determined using Terumo Mediace (blood sugar measurementset), and a rat with at least 400 mg/dl was selected from rats thustreated. STZ was dissolved in a citrate buffer (pH 4.5) and administeredwithin 5 minutes after it was dissolved.

For insulin-independent diabetes, insulin secretion-deficient typediabetes was generated by administering streptozotocin (STZ) to neonatalrats (“Saibokogaku”, Extra Issue, Medical Experiment Manual Series,Strategy for Study of Diabetes, edited by Susumu Kiyono and YoshikazuOka, published by Shushunsha, Japan).

STZ was subcutaneously administered at a dose of 90 mg/kg at 2 days old.At 4 days of age, blood was collected by cardiac puncture, and its bloodsugar level was determined using Terumo Mediace (blood sugar measurementset), and a rat with at least 275 mg/dl was selected from rats thustreated. STZ was dissolved in a citrate buffer (pH 4.5) and administeredwithin 5 minutes after it was dissolved.

(3) ¹³C breath test

A rat fasted overnight was anesthetized by intraperitonealadministration of Nembutal (50 mg/kg) and fixed as shown in FIG. 2.Blood was collected from the tail vein, and its sugar level wasdetermined using Terumo Mediace (blood sugar measurement set). 100 mg/kg¹³C-glucose or sodium ¹³C-pyruvate (0.1 g/ml) dissolved in physiologicalsaline was administered via the femoral vein, and the head was coveredwith a cylindrical tube, and its exhalation was sucked into a carbondioxide meter CAPSTAR-100 (CWE, Inc.). An exhalation was collected in anvolume of about 25 μl for each measurement through a Hamilton syringe(FIG. 2). The flow rate of the carbon dioxide meter was controlled suchthat its carbon dioxide level was within the range of 3.5±0.5%. The ¹³Clevel in exhaled CO₂ was determined in a gas chromatography massspectrophotometer (GC-MS). The analytical conditions for GC-MS are asfollows:

[GC-MS conditions] Apparatus: Shimadzu GC-MS QP-5000 [Shimadzu Co.,Ltd.]. Column: 0.32 mm × 25 m (ID × L) fused silica capilliary column.Ionization method: EI (electron impact) method. Gasification chambertemperature: 60° C. Column temperature: 60° C. GC interface temperature:230° C. Carrier gas: He. Carrier gas pressure: 20 Kpa. Measurement mode:SIM (selected ion monitoring). Measurement ions: m/z = 45, 46, 47.Sample injection volume: 20 μl.

¹³C-glucoses used were 1-¹³C-glucose (¹³C purity of carbon at the1-position: 99 atom-%, a product of EURISO-TOP Ltd. or CIL Ltd.),2-¹³C-glucose (¹³C purity of carbon at the 2-position: 99 atom-%, aproduct of ISOTEC Ltd.), 3-¹³C-glucose (¹³C purity of carbon at the3-position: 99 atom-%, a product of ICON Ltd.), and 6-¹³C-glucose (¹³Cpurity of carbon at the 6-position: 99 atom-%, a product of CIL Ltd. orICON Ltd.). ¹³C-pyruvic acid used was sodium 3-¹³C-pyruvate (¹³C purityof carbon at the 3-position: 99 atom-%, a product of ICON Ltd.). Therectum temperature was monitored through the experiment, and the bodytemperature was kept at 37° C. on a warming mat. After the experimentwas finished, whole blood was collected from the abdominal aorta andused as a sample for measurement of fructosamine levels in blood. Theanalysis of fructosamine was entrusted to BML Ltd. After collection ofblood, the rat used in the experiment was killed by administering anexcess anesthetic.

Method of Calculating ¹³C levels

The ratio of the presence of an oxygen isotope in a sample was assumedto be the ratio in the nature, and its ¹³C level was calculated from theion peak areas of m/z=45, 46 in the following formula. The ratio inareas of m/z=45, 46 (A45/A46) was assumed to be “a” according toJapanese Patent LOP Publication No. 120434/95.

¹³C level (%)={(0.004176−0.0007462a)/(0.9944396+0.0034298a)}×100  (Formula 1)

Δ¹³C (‰) Calculation Method

Calculated from ¹³C level in exhaled CO₂ (¹³C t min.) and ¹³C level inCO₂ standard gas (¹³C std) at each point in the following formula:

Δ¹³C level (‰)={(¹³C t min.−¹³C 0 min.)/¹³C std}×1000   (Formula 2)

(4) Measurement of insulin levels in blood

A by-path was formed by cannulation between the femoral artery andfemoral vein in a rat previously fasted overnight under anesthesia byintraperitoneal administration of Nembutal (50 mg/kg). The by-path wasprovided with a branch through which heparin (No. 15077-019, a productof GIBCO. BRL) was administered (100 U/rat). After ¹³C-glucose (0.1g/ml) dissolved in physiological saline was administered (100 mg/kg)through the branch, blood was collected with time and examined for bloodsugar levels and insulin levels. The insulin levels were determinedusing an insulin measurement kit (a product of Morinaga SeikagakuKenkyusho, Japan).

[2] Results

(1) 1-¹³C-glucose breath test {circle around (1)}

Animals examined were male Sprague-Dawley strain (SD) normal rats (four8-week-old rats and four 11-week-old rats), male SD rats withinsulin-independent diabetes (four 8-week-old rats and four 11-week-oldrats), and male SD rats with insulin-dependent diabetes (four 8-week-oldrats, four 9-week-old rats, and four 11-week-old rats; STZ wasadministered when the rats were 7-week-old). FIG. 3 shows the results ofthe measurement of degrees of increase of ¹³C levels in exhaled CO₂(Δ¹³C (‰)) at 20 minutes after intravenous injection of 100 mg/kg1-¹³C-glucose. FIG. 4 shows the results of the measurement of degrees ofincrease of ¹³C levels in exhaled CO₂ (Δ¹³C (‰)) at 20 minutes afterintravenous injection of 100 mg/kg 1-¹³C-glucose, versus blood sugarlevels just before administration of the glucose.

The distribution of AC levels at 20 minutes after administration (FIG.3) showed high levels over about 125 ‰ in the normal rats while lowlevels below about 125 ‰ in the rats with insulin-dependent diabetes andthe rats with insulin-independent diabetes. It is assumed that in thecase of higher sugar levels before administration, the degree ofdilution of administered 1-¹³C-glucose in blood is rendered higher, thusdecreasing the degree of discharge of ¹³C into an exhalation. Actually,it is observed that Δ¹³C levels decrease as blood sugar levels increases(FIG. 4). In the normal fasting blood sugar range (about 100 mg/dl),however, the Δ¹³C levels in the animals with diabetes are lower thanthose of the normal animals even although both of them have almost thesame fasting blood sugar levels (FIG. 4). Therefore, it can be said thatthe 1-¹³C-glucose breath test does not only mean the degree of dilutionof administered 1-¹³C-glucose, that is, blood sugar levels.

As exemplified above, the 1-¹³C-glucose breath test can distinguishbetween diabetes and normal in the same group with normal fasting bloodsugar levels, and can thus serve as an accurate and superior primaryscreening method.

(2) 1-¹³C-glucose breath test {circle around (2)}

Animals examined were male SD normal rats (five 11-week-old rats) andmale SD rats with insulin-independent diabetes (five 11-week-old rats).The 1-¹³C-glucose breath test and the measurement of insulin levels inblood were carried out in the same rats. 100 mg/kg of 1-¹³C-glucose wasadministered into the rats through the branch of the by-path providedbetween the femoral artery and femoral vein. Blood was collected beforeadministration and at 1, 2, 3, 5, 7, 10 and 15 minutes afteradministration, and insulin levels in blood were determined. FIG. 5shows the total amount of insulin secreted for the 15 minutes afteradministration versus the determined increase of ¹³C levels in exhaledCO₂.

Because the Δ¹³C levels at 20 minutes after administration of the1-¹³C-glucose is in good relation with the total amount of insulinsecreted for the first 15 minutes (FIG. 5), it is considered that thisbreath test is also useful as an examination method for determining acourse of action for therapy.

(3) 3-¹³C-glucose breath test {circle around (1)}

Animals examined were male SD normal rats (four 8-week-old rats and four11-week-old rats), male SD rats with insulin-independent diabetes (four8-week-old rats and four 11-week-old rats), and male SD rats withinsulin-dependent diabetes (four 8-week-old rats, four 9-week-old rats,and four 11-week-old rats; STZ was administered when the rats were7-week-old). FIG. 6 shows the results of increase of ¹³C levels inexhaled CO₂ (Δ¹³C (‰)) at 20 minutes after intravenous injection of 100mg/kg 3-¹³C-glucose, versus fasting blood sugar levels beforeadministration of the glucose.

The distribution of Δ¹³C levels at 20 minutes after administration (FIG.6) indicates that owing to the influence of blood sugar levels beforeadministration, Δ¹³C levels tend to decrease as blood sugar levelsincrease, as is the case with the 1-¹³C-glucose breath test. However,insulin-dependent diabetes group tends to show lower Δ¹³C levelscompared with insulin-independent group, in spite of similar fastingblood sugar levels between both groups. Therefore, this breath test isconsidered usable for the diagnosis of the type of diabetes incombination with the measurement of fasting blood sugar levels.

(4) 3-¹³C-glucose breath test {circle around (2)}

Animals examined were male SD normal rats (four 11-week-old rats), maleSD rats with insulin-independent diabetes (six 11-week-old rats), andmale SD rats with insulin-dependent diabetes (four 11-week-old rats; STZwas administered when the rats were 9-week-old).

FIG. 7 shows the results of increase of ¹³C levels in exhaled CO₂ (Δ¹³C(‰)) at 5, 10, 15 and 20 minutes after intravenous injection of 100mg/kg 3-¹³C-glucose. In the curves of ΔC for the first 20 minutes afteradministration of 3-¹³C-glucose (FIG. 7), the slope of the curve fromthe rats with insulin-independent diabetes tends to decrease in thelater half. The curves at 10 to 20 minutes after administration indicatethe following tendency of the slopes: normal rats>rats withinsulin-independent diabetes>rats with insulin-dependent diabetes (FIG.8). Therefore, 3-¹³C-glucose breath test can be used for both diagnosisof diabetes and diagnosis of the type of diabetes.

(5) 3-¹³C-glucose breath test {circle around (3)}

Animals examined were male SD normal rats (four 8-week-old rats and four11-week-old rats), male SD rats with insulin-independent diabetes (four8-week-old rats and four 11-week-old rats), and male SD rats withinsulin-dependent diabetes (four 8-week-old rats, four 9-week-old ratsand four 11-week-old rats; STZ was administered when the rats were7-week-old). FIG. 9 shows the results of increase of ¹³C levels inexhaled CO₂ (Δ¹³C (‰)) and fructosamine levels in blood at 20 minutesafter intravenous administration of 100 mg/kg 3-¹³C-glucose.

When Δ¹³C levels and fructosamine levels at 20 minutes afteradministration are plotted, Δ¹³C levels in the rats withinsulin-dependent diabetes were lower than those in the rats withinsulin-independent diabetes even though both of them have similarfructosamine levels (FIG. 9). Many reports have revealed that patientswith symptoms of insulin-dependent diabetes at a first stage of theonset undergo transition to insulin-independent diabetes at a laterstage. Depending on such change in symptoms, it is necessary to altermethods such as insulin treatment etc., but conventional tests fordetermining only average blood sugar levels in terms of fructosamine,HbAIC etc. can miss such change. Accordingly, the 3-¹³C-glucose breathtest is also useful as a test for knowing such change in symptoms.

(6) 2-¹³C-glucose and 6-¹³C-glucose breath tests

Animals examined were male SD normal rats (two 8-week-old rats), male SDrats with insulin-independent diabetes (two 8-week-old rats), and maleSD rats with insulin-dependent diabetes (two 8-week-old rats). FIG. 10shows the time course of ¹³C levels in exhaled CO₂ (Δ¹³C (‰)) for 40minutes after intravenous injection of 2-¹³C-glucose. FIG. 11 shows thetime course of ¹³C levels in exhaled CO₂ (Δ¹³C (‰)) for 20 minutes afterintravenous injection of 6-¹³C-glucose. Both of the 2-¹³C-glucose breathtest (FIG. 10) and 6-¹³C-glucose breath test (FIG. 11) show that Δ¹³Clevels in the rats with insulin-dependent diabetes are lower than thosein the rats with insulin-independent diabetes, so both of the breathtests can be considered usable in diagnosis of the type of diabetes.

(7) 3-¹³C-pyruvate breath test

Animals examined were male Sprague-Dawley strain (SD) normal rats (four8-week-old rats and four 11-week-old rats), male SD rats withinsulin-independent diabetes (four 8-week-old rats and four 11-week-oldrats), and male SD rats with insulin-dependent-diabetes (four 8-week-oldrats, four 9-week-old rats and four 11-week-old rats; STZ wasadministered when the rats were 7-week-old). FIG. 12 shows the resultsof an increase of ¹³C levels in exhaled CO₂ (Δ¹³C (‰)) for 10 minutesafter intravenous injection of 100 mg/kg sodium 3-¹³C-pyruvate andfasting blood sugar levels in the same rats just before administrationof the pyruvate.

Δ¹³C levels in the rats with insulin-dependent diabetes that maintainhigh fasting blood sugar levels (blood sugar levels of not less than 200mg/dl) are distributed in the wide range from 100 to 600 (‰). In thenormal fasting blood sugar range of about 100 mg/dl, however, the normalrats show lower levels than about 400 (‰) while the rats withinsulin-independent diabetes and the rats with insulin-dependentdiabetes with fasting blood sugar levels of not more than 200 mg/dl showhigher levels than about 400 (‰).

The fasting blood sugar test used for the primary screening in diagnosisof diabetes is considered to miss about ⅔ of patients with diabetesbecause their blood sugar levels are in the normal range. However, thepresent 3-¹³C-pyruvate exhalation test can distinguish between memberswith diabetes and normal members in the same group having normal fastingblood sugar levels, and can thus serve as an accurate and superiorprimary screening method.

Pharmaceutical Preparation Example 1 (Injection)

10 parts by weight of 1-¹³C-glucose was dissolved in 90 parts by weightof physiological saline and sterilized by filtration through a Milliporefilter. The filtrate was introduced into a vial and sealed to give aninjection.

Pharmaceutical Preparation Example 2 (Internal Liquid Agent)

10 parts by weight of 1-¹³C-glucose was dissolved in 90 parts by weightof de-ionized water and sterilized by filtration through a Milliporefilter. The filtrate was introduced into a vial and sealed to give aninternal liquid agent..

Pharmaceutical Preparation Example 3 (Injection)

10 parts by weight of sodium 3-¹³C-pyruvate was dissolved in 90 parts byweight of physiological saline and sterilized by filtration through aMillipore filter. The filtrate was introduced into a vial and sealed togive an injection.

Pharmaceutical Preparation Example 4 (Internal Liquid Agent)

10 parts by weight of sodium 3-¹³C-pyruvate was dissolved in 90 parts byweight of de-ionized water and sterilized by filtration through aMillipore filter. The filtrate was introduced into a vial and sealed togive an internal liquid agent.

What is claimed is:
 1. A method for detecting a diabetic condition in asubject comprising: a) administering to said subject an effective amountof pyruvic acid labeled with ¹³C at a specific position; and b)measuring a level of exhaled labeled CO₂, wherein a reduced level ofexhaled labeled CO₂ compared to normal is indicative of an increasedlikelihood of said subject having or suffering from said diabeticcondition.
 2. The method according to claim 1, wherein said pyruvic acidlabeled with ¹³C at a specific position is selected from the groupconsisting of 1-¹³C-pyruvic acid, 2-¹³C-pyruvic acid and 3-¹³C-pyruvicacid.